The actin cytoskeleton of human lymphocytes plays a critical role in its ability to participate in host defense. Stimulation of T-cells with various agonists including antibodies to the T-cell receptor causes a rapid transient increase in filamentous actin (F-actin). Blocking this F-actin increase with cytochalasin not only blocks the capping of surface receptors but also blocks the release of IL2 and increase in DNA synthesis. Phosphoinositides are involved in the regulation of the T-cell cytoskeleton at several levels including the regulation of actin binding, capping, and severing proteins, as well as small GTPases. The goal of this application is to understand the role of the phospholipid-interacting adaptor proteins, pleckstrin-1 and pleckstrin 2, in the early events of lymphocyte cytoskeletal organization. The PKC substrate, pleckstrin-l, contains the two prototypic phospholipid-binding Pleckstrin Homology (PH) domains. We have shown that expressed pleckstrin, and its non-phosphorylatable paralog pleckstrin 2, induce actin reorganization through a process that is dependent on small molecular weight GTPases, such as Rac, integrins, and phospholipid kinases such as phosphatidylinositol 3-kinase (PI3K). Although, pleckstrin-1 is one of the most abundant proteins within lymphocytes (about 1 percent of total cellular protein), its in vivo role in these cells remains uninvestigated. We hypothesize that in lymphocytes, pleckstrin-1 and pleckstrin-2 modulate phospholipid second messengers and actin reorganization. To test this hypothesis, we propose a series of complementary studies, which utilize in vitro biochemical studies and tissue culture systems, as well as in vivo experiments with murine model systems. Together, these will allow us to determine: 1) how pleckstrin isoforms contribute to the lymphocyte cytoskeletal changes, 2) the consequences of targeted disruption of both pleckstrin-1 and pleckstrin-2 in lymphocyte signaling, and 3) the role of PI3K in lymphocyte cytoskeletal organization?